| Phosphotyrosine interaction domain containing 1 (PIDl) is a recently discovered gene upregulated in omantal adipose tissue of obese subjects. The gene has been shown to play a role in insulin resistance and lipid metabolism. In porcine longissimus dorsi muscle, its expression was posotively correlated with intramuscular fat (IMF) content, suggesting PID1 may be a candidate gene for IMF deposition. This study aimed at revealing the role of PID1 in intramuscular preadipocytes proliferation and differentiation. The major contents and findings of this study are as follows:1. Prokaryotic expression, purification and, polyclonal antibody preparation and tissue distribution of porcine PIDl geneThe CDS region of porcine PID1 gene was inserted to pET28a(+) vector, in which the protein was fuse-expressed with His6-tag at its N-terminus. In E. coli BL21(DE3), the recombinant protein could be efficiently induced (60% of tatal cellular proteins) with 0.1 mmol/L IPTG at 30℃ for 4 h. However, the recombinant protein was mainly in forms of inclusion bodies. An unfolding affinity chromatography approach was applied to extract and purify the protein. Since molecular mass of the recombinant protein in SDS-PAGE was approximate 3 kDa larger than its theoretic value, the protein was further identified by mass spectrometry. For biological activity assay, the purified protein was refolded by dylysis against gradient concentrations of urea and then its effect on proliferation of 3T3-L1 preadipocytes was investigated. The result showed that the recombinant protein promoted 3T3-L1 preadipocytes proliferation, suggesting recombinant porcine PID1 protein prepared in this study was biologically active.Polyclonal antibodies were raised by immunizing Sprague-Dawley (SD) rats using the purified protein. Antibody titer of the polyclonal antibodies was approximately 1:20480 in ELISA and the polyclonal antibodies could specially identify recombinant porcine PID1 protein in western blot. This suggested the polyclonal antibodies made in this experiment can be used for further studies on PID1 gene.Tussue expression profile of PID1 in DLY pig was analyzed using the polyclonal antibodies. Immunoblot assay indicatied that the PID1 proteins were mainly present in skeletal muscles such as extensor digitorum longus, soleus, longissimus dorsi muscle and psoas major, a relatively lower expression was seen in liver. This suggested that PID1 may play an important role in lipid metabolism, especially the lipid metabolism in skeletal muscle.2. Effect of PIDl on porcine intramuscular preadipocyte proliferation and differentiationSince mammal intramuscular triglycerides (at least 80%) were mainly stored in intramuscluar adipocytes, the effects of PID1 on porcine intramuscular preadipocytes proliferation and differentiation were investigated. Primary intramuscular preadipocytes were isolated from 3-day pig longissimus dorsi muscle by the differential velocity adherent technique. Nearly 100% of the cells expressed Pref-1 as was shown by immunofluorescence assay. And the cells were able to differentiate into mature adipocytes by Oil red O staining. To determine whether PID1 affects porcine intramuscular preadipocyte proliferation, two experiments were designed. First, primary porcine preadipocytes were pretreated with recombinant porcine PID1 proteins or equal volume of phosphate buffered saline (PBS). At the same time, the cells were transfected with plasmid pcDNA3.1(+)-pPID1 or empty vector. Twenty-four hours later, the porcine PID1 significantly promoted porcine intramuscular preadipocytes proliferation when compared with the control group. To investigate the role of PIDl in porcine intramuscular preadipocyte differentiation, the cells were transfected with plasmid pcDNA3.1(+)-pPID1 or empty vector. The cells were cultured in differentiation medium for 8 days. The mRNA levels of the adipocyte differentiation transcription factors peroxisome proliferators-activated receptor y (PPARy), CCAAT/enhancer binding protein a (C/EBPa), and lipoprotein lipase (LPL) were significantly increased. Western blot analysis also showed that the protein levels of PPARy and C/EBPa were significantly upregulated after transfection with porcine PIDl.In summary, the paper first prepared biologically active recombinant porcine PID1 protein and its polyclonal antibody. PID1 tissue expression profile in pig analyzed by the polyclonal antibodies showed that PID1 were mainly present in skeletal muscles and liver, suggesting a role of PID1 in regulation of adipogenesis within skeletal muscle. In addition, the present study provided the first evidence that PID1 promote porcine intramuscular preadipocyte proliferation and differentiation. |
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